Lantibiotics are ribosomally produced, post translationally modified peptide natural products with antibiotic activity. Many exhibit unique modes of action by targeting highly conserved steps in bacterial peptidoglycan synthesis and/or disrupting the cell wall and plasma membrane. Given the importance of these structures to cellular viability, it is noteworthy that lantibiotics appear to elude the typical resistance pathwys. Thus, they remain potent against pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Due to the increasing threat of antibacterial resistance to human health, lantibiotics have been receiving increased consideration as potential treatments for infection. Lantibiotics are characterized by the presence of lanthionine (Lan) and methyllanthionine (MeLan) rings. These unusual structural features arise from the posttranslational, enzymatic modification of precursor peptides. Enzymatic dehydration of serine and threonine residues present in the precursor peptide yield dehydroalanine and dehydrobutyrine moieties, respectively. Michael-type addition of cysteine residues to the dehydrated residues produces the distinctive Lan and MeLan rings. In some systems a single bifunctional enzyme (LanM) is responsible for both dehydration and cyclization. The brevity of the genetically encoded biosynthetic pathways provides a remarkable opportunity for reengineering the lantibiotic machinery to generate compounds with improved therapeutic potential. Facilitated by the increasing availability of genomic information, there has been an explosion of newly discovered lantibiotic gene clusters. One of the more intriguing findings is a class of two-component lantibiotics that segregate cellular targeting from antimicrobial activity. For example, the two units of haloduracin (Hal and Hal) act in synergy to achieve nanomolar activity against a range of Gram-positive organisms. Hal is proposed to bind the peptidoglycan precursor lipid II, leading to the recruitment of Hal, pore formation and membrane disruption. However, molecular details about the interactions that lead to the potent bioactivity of haloduracin are lacking. Thus, the long-term goal of this proposal is to establish te mode of action of two-component lantibiotics and to use that knowledge to generate improved lantibiotics. To this end, the LanM enzymes that produce haloduracin will be engineered to generate constitutively active lantibiotic synthetases capable of producing analogues from chemically synthesized linear peptides. This methodology will allow the incorporation of non-natural features that will expand the chemical space beyond that accessible to Nature. Wild-type haloduracin and new analogues will be evaluated to uncover chemical features that enhance (or degrade) binding between haloduracin and its targets. The results of this work will enhance the mechanistic understanding of lantibiotic bioactivity, which may guide the development of new antibacterial compounds.